嗜伊紅血球陽離子蛋白(eosinophil cationic protein, ECP) 是由人類嗜伊紅血球釋放到細胞間質的抗病原菌顆粒蛋白。ECP與另一顆粒蛋白─嗜伊紅血球神經毒素(eosinophil derived neurotoxin, EDN) 於氣喘病人中大量表現，因此為臨床檢驗患者過敏嚴重度的生物標誌。ECP和EDN都屬於人類核醣核酸水解酶A家族(RNase A family) 的一員，並能進入特定細胞中。ECP能結合細胞表面的氨基葡聚醣，特別是硫酸乙醯肝素醣蛋白，再經胞飲作用進入支氣管上皮細胞。通常能與肝素/硫酸乙醯肝素結合的胺基酸序列為帶正電及疏水性胺基酸，本研究之目的為找出ECP與肝素/硫酸乙醯肝素的結合區位。將ECP之精胺酸(R)、色胺酸(W)和離胺酸(K)分別點突變為丙胺酸(A)，細胞表面酵素連結免疫吸附分析(cell ELISA)顯示R22A, R34A, R36A, K38A, R45A, R73A, R77A突變株ECP之結合細胞表面的能力大幅下降。進一步發現ECP之主要的肝素/硫酸乙醯肝素結合區位為34RWRCK38，與一般認知的結合區位樣式XBBXXB不同。除此之外，利用不同肝素衍生物來做競爭試驗，發現肝素上2-O-sulfation位置對於與嗜伊紅血球陽離子蛋白結合較為重要。另一方面，具有ECP上的肝素結合區位的胜肽會隨著時間與濃度的增加而增加其結合上人類肺細胞株的數量。本研究直接證明ECP和肝素之間的生物功能與分子作用。

Eosinophil cationic protein (ECP) is degranulated by eosinophils and released into the extracellular space to defense against pathogens. With another eosinophil granule protein, eosinophil derived neurotoxin (EDN), high level of ECP and EDN expression has been detected in asthma patients, and thus considered as a biomarker for diagnosis of disease severity. Both of ECP and EDN belong to human ribonuclease A (RNase A) superfamily and are able to enter several cells. Endocytosis of ECP into bronchial epithelial cells was greatly dependent on the cell surface glycosaminoglycan (GAGs), specifically heparan sulfate proteoglycans (HSPGs). The present study focuses on identification and characterization of the essential heparin/heparan sulfate (HS) binding motifs on ECP. HS binding motif has been reported to be mainly correlated with positive charge and aromatic residues, so each of the arginine (R), trytophan (W) and lysine (K) residues on ECP was specifically mutated to alanine, and the cell surface binding activity of mutant ECP was evaluated employing cell-enzyme-linked immunosorbent assay (ELISA). It was found that ECP-R22A, R34A, R36A, K38A, R45A, R73A and R77A showed significantly decreased HS binding activity, and the major heparin/HS binding motif in ECP was located at 34RWRCK38. The sequence composition and pattern of this motif were different from those of conventional heparan binding motifs (XBBXXB). In addition, de-2-O-sulfated heparin could not inhibit the cell binding ability of ECP as well as other heparin derivatives, indicating that ECP had high affinity to 2-O-sulfation of heparin. On the other hands, heparin binding peptide (ECP32-41) could bind BEAS-2B cells in a time- and concentration-dependent manner. This discovery provided the first direct evidence in biological functions and molecular interactions between ECP and heparin sulfate.

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